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Objectives: CpG ODN is a Toll-like receptor 9 agonist with immunotherapeutic potential for many cancer types, including aggressive breast cancers. There is strong interest in utilizing CpG ODN as an adjuvant to improve clinical efficacy of current treatments and immunogenicity of breast cancers not traditionally responsive to active immunotherapy, such as those that are human epidermal growth factor receptor 2 (HER2)-positive. This study aimed to study the efficacy and safety of combination CpG ODN plus anti-HER2 antibody trastuzumab treatment in patients with advanced/metastatic breast cancer., Methods: This single-arm, open-label phase II clinical trial treated patients (n = 6) with advanced/metastatic HER2-positive breast cancer with weekly subcutaneous CpG ODN and trastuzumab. Patients may have received any number of prior therapies to be enrolled (most enrolled at median 1 prior line of chemotherapy). Peripheral blood was collected at baseline and weeks 2, 6, 12, and 18 for immune analyses. Six patients were enrolled and 50% achieved stable disease (SD) response., Results: Median PFS was 8.3 months. Three of the six patients enrolled opted to stop treatment due to tolerability issues. Multiplex assay for cytokine measurements revealed significantly higher VEGF-D levels at week 2 compared to baseline. Peripheral blood mononuclear cells analyzed by flow cytometry showed a significant increase in monocytic MDSC between weeks 6 and 12. Patients with progressive disease tended to have higher levels of week 6 monocytic MDSC and PD-1+ T cells than patients with SD. NK cell populations did not significantly change throughout treatment., Conclusions: CpG ODN and trastuzumab treatment of metastatic HER2 + breast cancer was safe but was not tolerable for all patients. This combination did induce potentially predictive immune profile changes in treated patients with metastatic HER2 + breast cancer, the significance of which needs to be further explored., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Neonatal units across the world have altered their policies to prevent the spread of infection during the COVID-19 pandemic. Our aim was to report parental experience in two European neonatal units during the pandemic., Methods: Parents of infants admitted to each neonatal unit were asked to complete a questionnaire regarding their experience during the COVID-19 pandemic. At King's College Hospital, UK (KCH), data were collected prospectively between June 2020 and August 2020 (first wave). At the Hospital Clínic Barcelona (HCM), data were collected retrospectively from parents whose infants were admitted between September 2020 and February 2021 (second and third wave)., Results: A total of 74 questionnaires were completed (38 from KCH and 36 from HCM). The parents reported that they were fully involved or involved in the care of their infants in 34 (89.4%) responses in KCH and 33 (91.6%) responses in HCM. Quality time spent with infants during the pandemic was more negatively affected at KCH compared with HCM (n=24 (63.2%) vs n=12 (33.3%)). Parents felt either satisfied or very satisfied with the updates from the clinical care team in 30 (79.0%) responses at KCH and 30 (83.4%) responses in HCM. The parents felt that the restrictions negatively affected breast feeding in six (15.8%) responses at KCH and two (5.6%) responses in HCM. Travelling to the hospital was reported overall to be sometimes difficult (39.2%); this did not differ between the two units (14 (36.8%) respondents at KCH and 15 (41.6%) from HCM). Furthermore, the self-reported amount of time spent giving kangaroo care also did not differ between the two countries., Conclusion: Restrictive policies implemented due to the COVID-19 pandemic had a negative impact on the perception of quality of time spent by parents with their newborns admitted to neonatal units., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Poly(ADP-ribose) polymerase inhibitors (PARPis) are U.S. Food and Drug Administration (FDA) approved for treatment of BRCA-mutated metastatic breast cancer. Furthermore, the BROCADE studies demonstrated benefit of adding an oral PARPi, veliparib, to carboplatin and paclitaxel in patients with metastatic breast cancer harboring BRCA mutation. Given multiple possible dosing schedules and the potential benefit of this regimen for patients with defective DNA repair beyond BRCA, we sought to find the recommended phase II dose (RP2D) and schedule of veliparib in combination with carboplatin in patients with advanced breast cancer, either triple-negative (TNBC) or hormone receptor (HR)-positive, human epidermal growth receptor 2 (HER2) negative with defective Fanconi anemia (FA) DNA-repair pathway based on FA triple staining immunofluorescence assay., Materials and Methods: Patients received escalating doses of veliparib on a 7-, 14-, or 21-day schedule with carboplatin every 3 weeks. Patients underwent [18]fluoro-3'-deoxythymidine ( 18 FLT) positron emission tomography (PET) imaging., Results: Forty-four patients (39 TNBC, 5 HR positive/HER2 negative with a defective FA pathway) received a median of 5 cycles (range 1-36). Observed dose-limiting toxicities were grade (G) 4 thrombocytopenia (n = 4), G4 neutropenia (n = 1), and G3 akathisia (n = 1). Common grade 3-4 toxicities included thrombocytopenia, lymphopenia, neutropenia, anemia, and fatigue. Of the 43 patients evaluable for response, 18.6% achieved partial response and 48.8% had stable disease. Median progression-free survival was 18.3 weeks. RP2D of veliparib was established at 250 mg twice daily on days 1-21 along with carboplatin at area under the curve 5. Patients with partial response had a significant drop in maximum standard uptake value (SUV max ) of target lesions between baseline and early in cycle 1 based on 18 FLT-PET (day 7-21; p trend  = .006)., Conclusion: The combination of continuous dosing of veliparib and every-3-week carboplatin demonstrated activity and an acceptable toxicity profile. Decrease in SUV max on 18 FLT-PET scan during the first cycle of this therapy can identify patients who are likely to have a response., Implications for Practice: The BROCADE studies suggest that breast cancer patients with BRCA mutation benefit from addition of veliparib to carboplatin plus paclitaxel. This study demonstrates that a higher dose of veliparib is tolerable and active in combination with carboplatin alone. With growing interest in imaging-based early response assessment, the authors demonstrate that decrease in [18]fluoro-3'-deoxythymidine positron emission tomography (FLT-PET) SUV max during cycle 1 of therapy is associated with response. Collectively, this study established a safety profile of veliparib and carboplatin in advanced breast cancer while also providing additional data on the potential for FLT-PET imaging modality in monitoring therapy response., (© 2020 The Authors. The Oncologist published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Background: While combinations of immune checkpoint (ICP) inhibitors and neo-adjuvant chemotherapy (NAC) have begun testing in patients with breast cancer (BC), the effects of chemotherapy on ICP expression in circulating T cells and within the tumor microenvironment are still unclear. This information could help with the design of future clinical trials by permitting the selection of the most appropriate ICP inhibitors for incorporation into NAC., Methods: Peripheral blood samples and/or tumor specimens before and after NAC were obtained from 24 women with operable BC. The expression of CTLA4, PD-1, Lag3, OX40, and Tim3 on circulating T lymphocytes before and at the end of NAC were measured using flow cytometry. Furthermore, using multi-color immunohistochemistry (IHC), the expression of immune checkpoint molecules by stromal tumor-infiltrating lymphocytes (TILs), CD8+ T cells, and tumor cells was determined before and after NAC. Differences in the percentage of CD4+ and CD8+ T cells expressing various checkpoint receptors were determined by a paired Student's t-test., Results: This analysis showed decreased ICP expression by circulating CD4+ T cells after NAC, including significant decreases in CTLA4, Lag3, OX40, and PD-1 (all p values < 0.01). In comparison, circulating CD8+ T cells showed a significant increase in CTLA4, Lag3, and OX40 (all p values < 0.01). Within tumor samples, TILs, CD8+ T cells, and PD-L1/PD-1 expression decreased after NAC. Additionally, fewer tumor specimens were considered to be PD-L1/PD-1 positive post-NAC as compared to pre-NAC biopsy samples using a cutoff of 1% expression., Conclusions: This work revealed that NAC treatment can substantially downregulate CD4+ and upregulate CD8+ T cell ICP expression as well as deplete the amount of TILs and CD8+ T cells found in breast tumor samples. These findings provide a starting point to study the biological significance of these changes in BC patients., Trial Registration: NCT04022616.

Context: Walking football (soccer) has recently emerged as a physical activity option targeted at older males to enhance health and wellbeing., Design: This pilot study aimed to examine the feasibility of recruiting and retaining males aged 50 years and over to an 8-week walking football programme in a professional football club., Intervention: Participants were recruited via social media and assigned to an intervention group or a wait-list control group. The intervention group engaged in 1 h of walking football a week led by a community coach from the professional football club, followed by an optional social session in the club facility. Physiological and psychological outcome measures were obtained onsite at the football club facility (aiding compliance and retention) at baseline and following 8-weeks, from both groups. Semi-structured interviews were conducted after the 8-week programme and 1 year later, to explore motivations for engagement and the social impact., Results: The opportunity to engage in football and the link to a professional football club were key attractions. All participants recruited were overweight, sedentary, exhibited blood pressures outside normal ranges, and all but two were hypertensive. Adherence to the programme was 90% over 8 weeks, and of the participants who were contacted after one year, all (n = 6) had maintained engagement in walking football. Walking football is therefore a feasible, cost-effective method of recruiting and retaining males aged 50 years and over to a physical activity programme, though attrition is to be expected., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: In the UK, patients with one or two adenomas, of which at least one is ≥ 10 mm in size, or three or four small adenomas, are deemed to be at intermediate risk of colorectal cancer (CRC) and referred for surveillance colonoscopy 3 years post polypectomy. However, colonoscopy is costly, can cause discomfort and carries a small risk of complications., Objectives: To determine whether or not annual faecal immunochemical tests (FITs) are effective, acceptable and cost saving compared with colonoscopy surveillance for detecting CRC and advanced adenomas (AAs)., Design: Diagnostic accuracy study with health psychology assessment and economic evaluation., Setting: Participants were recruited from 30 January 2012 to 30 December 2013 within the Bowel Cancer Screening Programme in England., Participants: Men and women, aged 60-72 years, deemed to be at intermediate risk of CRC following adenoma removal after a positive guaiac faecal occult blood test were invited to participate. Invitees who consented and returned an analysable FIT were included., Intervention: We offered participants quantitative FITs at 1, 2 and 3 years post polypectomy. Participants testing positive with any FIT were referred for colonoscopy and not offered further FITs. Participants testing negative were offered colonoscopy at 3 years post polypectomy. Acceptibility of FIT was assessed using discussion groups, questionnaires and interviews., Main Outcome Measures: The primary outcome was 3-year sensitivity of an annual FIT versus colonoscopy at 3 years for detecting advanced colorectal neoplasia (ACN) (CRC and/or AA). Secondary outcomes included participants' surveillance preferences, and the incremental costs and cost-effectiveness of FIT versus colonoscopy surveillance., Results: Of 8008 invitees, 5946 (74.3%) consented and returned a round 1 FIT. FIT uptake in rounds 2 and 3 was 97.2% and 96.9%, respectively. With a threshold of 40 µg of haemoglobin (Hb)/g faeces (hereafter referred to as µg/g), positivity was 5.8% in round 1, declining to 4.1% in round 3. Over three rounds, 69.2% (18/26) of participants with CRC, 34.3% (152/443) with AAs and 35.6% (165/463) with ACN tested positive at 40 µg/g. Sensitivity for CRC and AAs increased, whereas specificity decreased, with lower thresholds and multiple rounds. At 40 µg/g, sensitivity and specificity of the first FIT for CRC were 30.8% and 93.9%, respectively. The programme sensitivity and specificity of three rounds at 10 µg/g were 84.6% and 70.8%, respectively. Participants' preferred surveillance strategy was 3-yearly colonoscopy plus annual FITs (57.9%), followed by annual FITs with colonoscopy in positive cases (31.5%). FIT with colonoscopy in positive cases was cheaper than 3-yearly colonoscopy (£2,633,382), varying from £485,236 (40 µg/g) to £956,602 (10 µg/g). Over 3 years, FIT surveillance could miss 291 AAs and eight CRCs using a threshold of 40 µg/g, or 189 AAs and four CRCs using a threshold of 10 µg/g., Conclusions: Annual low-threshold FIT with colonoscopy in positive cases achieved high sensitivity for CRC and would be cost saving compared with 3-yearly colonoscopy. However, at higher thresholds, this strategy could miss 15-30% of CRCs and 40-70% of AAs. Most participants preferred annual FITs plus 3-yearly colonoscopy. Further research is needed to define a clear role for FITs in surveillance., Future Work: Evaluate the impact of ACN missed by FITs on quality-adjusted life-years., Trial Registration: Current Controlled Trials ISRCTN18040196., Funding: National Institute for Health Research (NIHR) Health Technology Assessment programme, NIHR Imperial Biomedical Research Centre and the Bobby Moore Fund for Cancer Research UK. MAST Group Ltd provided FIT kits., Competing Interests: Wendy Atkin and Amanda J Cross report grants from the National Institute for Health Research (NIHR) Health Technology Assessment (HTA) programme, grants from Cancer Research UK (Population Research Committee – Programme Award C8171/A16894) and non-financial support from Eiken Chemical Co. Ltd (Tokyo, Japan) (MAST is UK distributor) during the conduct of the study. Stephen Morris is a member of the NIHR Health Services and Delivery Research funding board. Sheena Pearson, Carolyn Piggott and Julia Snowball all report grants from the NIHR HTA programme during the conduct of the study.

New Zealand (NZ) is a small country with an export-led economy with above 90% of primary production exported. Plant-based primary commodities derived from the pastoral, horticultural and forestry sectors account for around half of the export earnings. Productivity is characterized by a history of innovation and the early adoption of advanced technologies. Gene editing has the potential to revolutionize breeding programmes, particularly in NZ. Here, perennials such as tree crops and forestry species are key components of the primary production value chain but are challenging for conventional breeding and only recently domesticated. Uncertainty over the global regulatory status of gene editing products is a barrier to invest in and apply editing techniques in plant breeding. NZs major trading partners including Europe, Asia and Australia are currently evaluating the regulatory status of these technologies and have not made definitive decisions. NZ is one of the few countries where the regulatory status of gene editing has been clarified. In 2014, the NZ Environmental Protection Authority ruled that plants produced via gene editing methods, where no foreign DNA remained in the edited plant, would not be regulated as GMOs. However, following a challenge in the High Court, this decision was overturned such that NZ currently controls all products of gene editing as GMOs. Here, we illustrate the potential benefits of integrating gene editing into plant breeding programmes using targets and traits with application in NZ. The regulatory process which led to gene editing's current GMO classification in NZ is described and the importance of globally harmonized regulations, particularly to small export-driven nations is discussed.

Background: To assess public preferences for colorectal cancer (CRC) surveillance tests for intermediate-risk adenomas, using a hypothetical scenario., Methods: Adults aged 45-54 years without CRC were identified from three General Practices in England (two in Cumbria, one in London). A postal survey was carried out during a separate study on preferences for different first-line CRC screening modalities (non- or full-laxative computed tomographic colonography, flexible sigmoidoscopy, or colonoscopy). Individuals were allocated at random to receive a pack containing information on one first-line test, and a paragraph describing CRC surveillance recommendations for people who are diagnosed with intermediate-risk adenomas during screening. All participants received a description of two surveillance options: annual single-sample, home-based stool testing (consistent with Faecal Immunochemical Tests; FIT) or triennial colonoscopy. Invitees were asked to imagine they had been diagnosed with intermediate-risk adenomas, and then complete a questionnaire on their surveillance preferences., Results: 22.1 % (686/3,100) questionnaires were returned. 491 (15.8 %) were eligible for analysis. The majority of participants stated a surveillance preference for the stool test over colonoscopy (60.8 % vs 31.0 %; no preference: 8.1 %; no surveillance: 0.2 %). Women were more likely to prefer the stool test than men (66.7 % vs. 53.6 %; p = .011). The primary reason for preferring the stool test was that it would be done more frequently. The main reason to prefer colonoscopy was its superiority at finding polyps., Conclusions: A majority of participants stated a preference for a surveillance test resembling FIT over colonoscopy. Future research should test whether this translates to greater adherence in a real surveillance setting., Trial Registration: International Standard Randomised Controlled Trial Number registry, ISRCTN85697880 , prospectively registered on 25/04/2013.

Background: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy., Methods: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system., Results: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in < 25%. No sequence had resistance to all currently available drugs., Conclusion: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.

Background: The prevalence and the origin of HIV-1 subtype B, the most prevalent circulating clade among the long-term residents in Europe, have been studied extensively. However the spatial diffusion of the epidemic from the perspective of the virus has not previously been traced., Results: In the current study we inferred the migration history of HIV-1 subtype B by way of a phylogeography of viral sequences sampled from 16 European countries and Israel. Migration events were inferred from viral phylogenies by character reconstruction using parsimony. With regard to the spatial dispersal of the HIV subtype B sequences across viral phylogenies, in most of the countries in Europe the epidemic was introduced by multiple sources and subsequently spread within local networks. Poland provides an exception where most of the infections were the result of a single point introduction. According to the significant migratory pathways, we show that there are considerable differences across Europe. Specifically, Greece, Portugal, Serbia and Spain, provide sources shedding HIV-1; Austria, Belgium and Luxembourg, on the other hand, are migratory targets, while for Denmark, Germany, Italy, Israel, Norway, the Netherlands, Sweden, Switzerland and the UK we inferred significant bidirectional migration. For Poland no significant migratory pathways were inferred., Conclusion: Subtype B phylogeographies provide a new insight about the geographical distribution of viral lineages, as well as the significant pathways of virus dispersal across Europe, suggesting that intervention strategies should also address tourists, travellers and migrants.

Vitamin C (L-ascorbic acid, AsA) is an essential metabolite for plants and animals. Kiwifruit (Actinidia spp.) are a rich dietary source of AsA for humans. To understand AsA biosynthesis in kiwifruit, AsA levels and the relative expression of genes putatively involved in AsA biosynthesis, regeneration, and transport were correlated by quantitative polymerase chain reaction in leaves and during fruit development in four kiwifruit genotypes (three species; A. eriantha, A. chinensis, and A. deliciosa). During fruit development, fruit AsA concentration peaked between 4 and 6 weeks after anthesis with A. eriantha having 3-16-fold higher AsA than other genotypes. The rise in AsA concentration typically occurred close to the peak in expression of the L-galactose pathway biosynthetic genes, particularly the GDP-L-galactose guanyltransferase gene. The high concentration of AsA found in the fruit of A. eriantha is probably due to higher expression of the GDP-mannose-3',5'-epimerase and GDP-L-galactose guanyltransferase genes. Over-expression of the kiwifruit GDP-L-galactose guanyltransferase gene in Arabidopsis resulted in up to a 4-fold increase in AsA, while up to a 7-fold increase in AsA was observed in transient expression studies where both GDP-L-galactose guanyltransferase and GDP-mannose-3',5'-epimerase genes were co-expressed. These studies show the importance of GDP-L-galactose guanyltransferase as a rate-limiting step to AsA, and demonstrate how AsA can be significantly increased in plants.

Background: The genetic barrier, defined as the number of mutations required to overcome drug-selective pressure, is an important factor for the development of HIV drug resistance. Because of high variability between subtypes, particular HIV-1 subtypes could have different genetic barriers for drug resistance substitutions. This study compared the genetic barrier between subtypes using some 2000 HIV-1 sequences (>600 of non-B subtype) isolated from anti-retroviral-naive patients in Europe., Methods: The genetic barrier was calculated as the sum of transitions (scored as 1) and/or transversions (2.5) required for evolution to any major drug resistance substitution. In addition, the number of minor protease substitutions was determined for every subtype., Results: Few dissimilarities were found. An increased genetic barrier was calculated for I82A (subtypes C and G), V108I (subtype G), V118I (subtype G), Q151M (subtypes D and F), L210W (subtypes C, F, G, and CRF02&#95;AG), and P225H (subtype A) (P < 0.001 compared with subtype B). A decreased genetic barrier was found for I82T (subtypes C and G) and V106M (subtype C) (P < 0.001 vs subtype B). Conversely, minor protease substitutions differed extensively between subtypes., Conclusions: Based on the calculated genetic barrier, the rate of drug resistance development may be similar for different HIV-1 subtypes. Because of differences in minor protease substitutions, protease inhibitor resistance could be enhanced in particular subtypes once the relevant major substitutions are selected.

Background: Infection with drug-resistant human immunodeficiency virus type 1 (HIV-1) can impair the response to combination therapy. Widespread transmission of drug-resistant variants has the disturbing potential of limiting future therapy options and affecting the efficacy of postexposure prophylaxis., Methods: We determined the baseline rate of drug resistance in 2208 therapy-naive patients recently and chronically infected with HIV-1 from 19 European countries during 1996-2002., Results: In Europe, 1 of 10 antiretroviral-naive patients carried viruses with > or = 1 drug-resistance mutation. Recently infected patients harbored resistant variants more often than did chronically infected patients (13.5% vs. 8.7%; P=.006). Non-B viruses (30%) less frequently carried resistance mutations than did subtype B viruses (4.8% vs. 12.9%; P<.01). Baseline resistance increased over time in newly diagnosed cases of non-B infection: from 2.0% (1/49) in 1996-1998 to 8.2% (16/194) in 2000-2001., Conclusions: Drug-resistant variants are frequently present in both recently and chronically infected therapy-naive patients. Drug-resistant variants are most commonly seen in patients infected with subtype B virus, probably because of longer exposure of these viruses to drugs. However, an increase in baseline resistance in non-B viruses is observed. These data argue for testing all drug-naive patients and are of relevance when guidelines for management of postexposure prophylaxis and first-line therapy are updated.

Ascorbate is a critical compound in plants and animals. Humans are unable to synthesize ascorbate, and their main source of this essential vitamin are plants. However, the pathway of synthesis in plants is yet to be established, and several unknown enzymes are only postulated to exist. We describe a specific L-galactose-1-phosphate (L-gal-1-P) phosphatase that we partially purified from young kiwifruit (Actinidia deliciosa) berries. The enzyme had a native molecular mass of approximately 65 kDa, was completely dependent on Mg2+ for activity and was very specific in its ability to hydrolyze L-gal-1-P. The activity had a pH optimum of 7.0, a K(-M(L-gal-1-P) of 20-40 microM and a Ka(Mg2+) of 0.2 mM. The activity was inhibited by Mg2+ at concentrations >2 mM. The enzyme from Arabidopsis thaliana shoots showed similar properties to the kiwifruit enzyme. The Arabidopsis thaliana enzyme preparation was digested with trypsin, and proteins present were identified by using liquid chromatography-MS. One of 24 proteins present in our preparation was an Arabidopsis thaliana protein, At3g02870, annotated myo-inositol-1-phosphate phosphatase in GenBank, that matched the characteristics of the purified l-gal-1-phosphate phosphatase. We then expressed a kiwifruit homologue of this gene in Escherichia coli and found that it showed 14-fold higher maximum velocity for l-gal-1-P than myo-inositol-1-P. The expressed enzyme showed very similar properties to the enzyme purified from kiwifruit and Arabidopsis, except that its KM(L-gal-1-P) and Ka(Mg2+) were higher in the expressed enzyme. The data are discussed in terms of the pathway to ascorbate biosynthesis in plants.

An alpha-amylase gene product was isolated from apple fruit by reverse-transcriptase PCR using redundant primers, followed by 5' and 3' RACE. The gene is a member of a small gene family. It encodes a putative 46.9 kDa protein that is most similar to an alpha-amylase gene from potato (GenBank accession M79328). In apple fruit this new gene was expressed at low levels, as detected by reverse-transcriptase PCR, in a number of plant tissues and during fruit development. Highest levels of mRNA for this transcript were observed 3 to 9 days after placing apple fruit at 0.5 degrees C. Phylogenetic analysis of amino acid sequence places the potato and apple proteins as a distinct and separate new subgroup within the plant alpha-amylases, which appears to have diverged prior to the split between monocotyledonous and dicotyledonous plants. These two divergent alpha-amylases lack the standard signal peptide structures found in all other plant alpha-amylases, and have sequence differences within the B-domain and C-domain. However, comparisons with structures of known starch hydrolases suggest that these differences are unlikely to affect the enzymatic alpha-1,4-amylase function of the protein. This is the first report of upregulation of a dicotyledonous alpha-amylase in response to low temperature, and confirms the presence of a new family of alpha-amylases in plants.

The localization of pectin, cellulose, xyloglucan, and callose was compared in kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang and A. R. Ferguson var. deliciosa "Hayward") at harvest, at the end of the first phase of softening, and when ripe. Pectin was visualized using three different methods: labeling of galacturonic acid residues, labeling of negatively charged groups, and labeling with JIM 5 (nonesterified residues) and JIM 7 (methyl-esterified) monoclonal antibodies. Labeling of pectin gave different results depending on the detection system used. Differences related to patterns of change during ripening and to spatial distribution of label intensity. Cell wall pectin was available for labeling at all stages of fruit softening, but no clear differentiation of the middle lamella region was seen, although JIM 5 binding predominated where the middle lamellae joined the intercellular spaces in unripe fruit. Negatively charged groups (cationic gold labeling) and, to a lesser extent, galacturonic acid residues (Aplysia depilans gonad lectin labeling) were preferentially located near the cell wall/plasma membrane boundary. The lack of strong binding of the JIM antibodies indicated that the reactive groups were inaccessible. Cellulose remained intact and labeled densely across the wall at all stages of fruit ripening. Distribution of xyloglucan was patchy at harvest but was scattered throughout the wall later in ripening. Alterations to labeling of xyloglucan indicated that some epitopes were differentially exposed. Plasmodesmatal regions were clearly different in composition to other wall areas, showing an absence of cellulose labeling, specific pectin labeling, and callose presence. A similar predominance of pectin labeling compared with cellulose also occurred at the middle lamella wedge near intercellular spaces.

Mature fruit (kiwifruit) of Actinidia deliciosa var. deliciosa (A. Chev.), (C.F.) Liang and Ferguson cv. Haywood (Chinese gooseberry) were harvested and allowed to ripen in the dark at 20° C. Changes were recorded in metabolites, starch and sugars, adenine nucleotides, respiration, and sucrose and glycolytic enzymes during the initiation of starch degradation, net starch-to-sucrose conversion and the respiratory climacteric. The conversion of starch to sucrose was not accompanied by a consistent increase in hexose-phosphates, and UDP-glucose declined. The activity of sucrose phosphate synthase (SPS) measured with saturating substrate rose soon after harvesting and long before net sucrose synthesis commenced. The onset of sugar accumulation correlated with an increase in SPS activity measured with limiting substrates. Throughout ripening, until sucrose accumulation ceased, feeding [(14)C] glucose led to labelling of sucrose and fructose, providing evidence for a cycle of sucrose synthesis and degradation. It is suggested that activation of SPS, amplified by futile cycles, may regulate the conversion of starch to sugars. The respiratory climacteric was delayed, compared with net starchsugar interconversion, and was accompanied by a general decline of pyruvate and all the glycolytic intermediates except fructose-1,6-bisphosphate. The ATP/ ADP ratio was maintained or even increased. It is argued that the respiratory climacteric cannot be simply a consequence of increased availability of respiratory substrate during starch-sugar conversion, nor can it result from an increased demand for ATP during this process.

Cytoplasmic granules known to contain cationic arginine-rich proteins can be identified by the ammoniacal silver reaction (ASR) which provides a cytochemical marker detectable under the electron microscope. Only the large rod-shaped granules of the chicken polymorphonuclear leukocytes (heterophils) and the large spherical azurophilic granules of the rabbit neutrophilic polymorphonuclear leukocytes show the ASR product as a discrete particulate electron-dense deposit. The other smaller granules are devoid of reaction product, as are membranes and mitochondria. The intracellular localization of the ASR product, as are membranes and mitochondria. The intracellular localization of the ASR product on the large granules coincides with the ASR product localization on the same isolated granule populations, when the ammoniacal silver reaction is applied to these granules after their separation by sucrose-density gradients. The cationic proteins may have intraleukocytic bacteriolytic properties, since ASR product, presumably indicating cationic protein from discharged granules, appears to surround ingested bacteria within cytoplasmic phagosomes.

Pharyngeal muscle of the planarian Dugesia tigrina was studied by electron microscopy after osmium tetroxide fixation. The muscle cell was observed to contain one myofibril or bundle of myofilaments parallel to its longitudinal axis. The myofilaments were of two types, different in size and distribution. No Z lines or myofilament organization into cross or helical striations were seen. Dense bodies were seen as projections from an invagination of the plasma membrane and as dense lines parallel to the myofilaments. The muscle cells are surrounded by a plasma membrane which is structurally associated with dense body projections, with vesicles and cisternae of sarcoplasmic reticulum, and with synaptic nerve endings. The cell has sarcoplasmic projections perpendicular to its long axis; these projections are seen to contain the nucleus or mitochondria and granules. Mitochondria and granules are also seen in a sarcoplasm rim around the fibril. The dense bodies may serve as attachment for thin myofilaments and function in transmission of stimuli from plasma membrane to the interior of the fibril.

Two species of planarians were studied by fluorescence microscopy. Red fluorescence of uroporphyrin was observed localized in the epidermal rhabdites and subepidermal rhabdite-containing gland cells. Fluorescence was observed in isolated rhabdites of homogenates, but was not seen in rhabdites of the living animal. The identity of rhabdites was established by their location, shape, size, and acid-ophilic staining properties.